High-throughput flow cytometry compatible biosensor based on fluorogen activating protein technology

Authors

  • Yang Wu,

    Corresponding author
    1. UNM Center for Molecular Discovery, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131
    2. Department of Pathology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131
    • Department of Pathology, 915 Camino de Salud, IDTC Rm 2340, 1 University of New Mexico, MSC 09 5025, Albuquerque, NM 87131, USA
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  • Phillip H. Tapia,

    1. UNM Center for Molecular Discovery, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131
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  • Gregory W. Fisher,

    1. Carnegie Mellon University Technology Center of Networks and Pathways, Pittsburgh, Pennsylvania 15213
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  • Alan S. Waggoner,

    1. Carnegie Mellon University Technology Center of Networks and Pathways, Pittsburgh, Pennsylvania 15213
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  • Jonathan Jarvik,

    1. Carnegie Mellon University Technology Center of Networks and Pathways, Pittsburgh, Pennsylvania 15213
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  • Larry A. Sklar

    Corresponding author
    1. UNM Center for Molecular Discovery, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131
    2. Department of Pathology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131
    • Department of Pathology, 915 Camino de Salud, IDTC Rm 2340, 1 University of New Mexico, MSC 09 5025, Albuquerque, NM 87131, USA
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Abstract

Monitoring the trafficking of multiple proteins simultaneously in live cells is of great interest because many receptor proteins are found to function together with others in the same cell. However, existing fluorescent labeling techniques have restricted the mechanistic study of functional receptor pairs. We have expanded a hybrid system combining fluorogen-activating protein (FAP) technology and high-throughput flow cytometry to a new type of biosensor that is robust, sensitive, and versatile. This provides the opportunity to study multiple trafficking proteins in the same cell. Human beta2 adrenergic receptor (β2AR) fused with FAP AM2.2 and murine C-C chemokines receptor type 5 fused with FAP MG13 was chosen for our model system. The function of the receptor and the binding between MG13 and fluorogen MG-2p have been characterized by flow cytometry and confocal microscopy assays. The binding of fluorogen and the FAP pair is highly specific, while both FAP-tagged fusion proteins function similarly to their wild-type counterparts. The system has successfully served as a counter screen assay to eliminate false positive compounds identified in a screen against NIH Molecular Libraries Small Molecule Repository targeting regulators of the human β2AR. © 2013 International Society for Advancement of Cytometry

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